Fura-2 Signals Research Articles

An Antagonist of cADP-ribose Inhibits Arrhythmogenic Oscillations of Intracellular Ca2+ In Heart Cells

Oscillations of Ca2+ in heart cells are a major underlying cause of important cardiac arrhythmias, and it is known that Ca2+-induced release of Ca2+ from intracellular stores (the sarcoplasmic reticulum) is fundamental to the generation of such oscillations. There is now evidence that cADP-ribose may be an endogenous regulator of the Ca2+ release channel of the sarcoplasmic reticulum (the ryanodine receptor), raising the possibility that cADP-ribose may influence arrhythmogenic mechanisms in the heart. 8-Amino-cADP-ribose, an antagonist of cADP-ribose, suppressed oscillatory activity associated with overloading of intracellular Ca2+ stores in cardiac myocytes exposed to high doses of the beta-adrenoreceptor agonist isoproterenol or the Na+/K+-ATPase inhibitor ouabain. The oscillations suppressed by 8-amino-cADP-ribose included intracellular Ca2+ waves, spontaneous action potentials, after-depolarizations, and transient inward currents. Another antagonist of cADP-ribose, 8-bromo-cADP-ribose, was also effective in suppressing isoproterenol-induced oscillatory activity. Furthermore, in the presence of ouabain under conditions in which there was no arrhythmogenesis, exogenous cADP-ribose was found to be capable of triggering spontaneous contractile and electrical activity. Because enzymatic machinery for regulating the cytosolic cADP-ribose concentration is present within the cell, we propose that 8-amino-cADP-ribose and 8-bromo-cADP-ribose suppress cytosolic Ca2+ oscillations by antagonism of endogenous cADP-ribose, which sensitizes the Ca2+ release channels of the sarcoplasmic reticulum to Ca2+.

Calibration of Fura-2 signals introduces errors into measurement of thrombin-stimulated calcium mobilisation in human platelets

The intracellular calcium indicator dye Fura-2 has been widely used for the study of human platelet thrombin receptor-mediated calcium mobilisation in disease states. In general, authors (a) use a Fura-2/AM concentration of 2–3 μmol/l and (b) calibrate fluorescence signals on the basis of maximum and minimum ratios of fluorescence ( R max and R min) and the ratio of the calcium-free and calcium-bound fluorescence at an excitation wavelength of 380 nm ( “C 2/B 2” ). In the present study, it is found (a) that a greater peak response to thrombin is seen when 1 μmol/l Fura-2/AM rather than 2 or 3 μmol/l is used; and (b) that calibration leads to a poorer test-retest reliability and in general a greater variability of the obtained calcium signal than when the simple measurement of the 340 nm/380 nm fluorescence ratio is used. It is suggested that this poor variability is due to the presence of an extracellular factor that can quench the Fura-2 signal once the platelets have been permeabilised by detergent treatment. Consistent with this, addition of bovine serum albumin to the assay medium has no significant effect on the fluorescence ratio response to thrombin, but greatly increases the observed calibrated calcium signal.

Lead reduces depolarization-induced calcium entry in cultured DRG neurons without crossing the cell membrane: fura-2 measurements.

1. Cultured dorsal root ganglion of rat pups were depolarized by exposure to 50 mM K+ and the rise of [Ca2+]i was measured using fura-2 as an indicator. 2. Lead in the extracellular solution reduced the rise of [Ca2+]i in a concentration-dependent manner, with a threshold concentration of 0.25 microM. More than 80% of the calcium entry was prevented by approximately 5 microM lead. The IC50 and the Hill coefficient were 3.1 microM and 1, respectively. 3. This effect was considered to be due to a reduction of VACCCs, since applications of NMDA did not result in any rise of [Ca2+]i. 4. Since Pb2+ itself changes the fura-2 signal in a typical and characteristic manner, fura-2 is also an indicator for Pb2+. No changes in fura-2 signals were detected when lead (5 microM) was applied for several minutes in the absence of calcium, indicating that Pb2+ did not enter the cells. 5. Thus it is concluded that lead prevents calcium entry by reducing VACCCs but does not cross the cell membrane itself.

Effect of lidocaine on histamine release and Ca2+ mobilization from mast cells and basophils.

Various anesthetic drugs have been known to induce allergic reactions, which have been caused by histamine release from mast cells/basophils. Although lidocaine is reported to suppress allergic reactions, there have been no reports about lidocaine's direct effects to inhibit histamine release from mast cells/basophils. We examined the effect of lidocaine on histamine release in vitro from freshly extracted as well as cultured mast cells and basophils. Additionally, the effects of lidocaine on intracellular calcium concentration were monitored by assessing Fura-2 signals in cultured cells. Lidocaine (10(-3)-10(-2) M: approximately 234-2340 micrograms/ ml) inhibited both the IgE-dependent and IgE-independent histamine release from all mast cells/basophils in a dose-dependent manner. However, lidocaine inhibited the IgE-dependent response more than the IgE-independent response (P < 0.01). Lidocaine also inhibited increases in intracellular Ca2+ to a greater extent after IgE-dependent stimulation as compared with IgE-independent stimulation. The degree of the inhibition of histamine release by lidocaine appeared to parallel decreases in Ca2+ mobilization. Our results indicate that lidocaine directly inhibits histamine release from both rodent mast cells and human basophils in vitro at concentrations from 10(-3) to 10(-2) M (234 to 2340 micrograms/ml). That may be influenced by Ca2+ mobilization. Although these results are not immediately relevant to clinical practice, allergic reaction caused by direct effect of lidocaine seems to be impossible.

Relaxant actions of isoprenaline on guinea-pig isolated tracheal smooth muscle.

1. The effects of isoprenaline on membrane potential and intracellular Ca2+ concentration ([Ca2+]i) in guinea-pig isolated tracheal muscle were studied by use of intracellular micro-electrodes and fura-2 signals respectively. Measurements of membrane potential were carried out in the presence of spontaneously-generated muscle tone, whereas fura-2 signals were measured during contraction produced by exogenous prostaglandin E2 (100 nM). The potency of isoprenaline in causing relaxation was the same in these two different situations. 2. Isoprenaline (0.01 microM) produced relaxation accompanied by 5 mV hyperpolarization. A combination of tetraethylammonium (TEA, 10 mM) and verapamil (3 microM) did not alter the effects of isoprenaline. Removal of external K+ did not increase the degree of hyperpolarization produced by isoprenaline. 3. In the presence of TEA (10 mM) and verapamil (3 microM), isoprenaline (0.03-1 microM) reduced [Ca2+]i concentration-dependently. A similar degree of inhibition was observed when isoprenaline was applied during the maintained contraction induced by prostaglandin E2 and against the contraction evoked by the addition of Ca2+ to tissues bathed in a Ca(2+)-free medium and pretreated with both isoprenaline and prostaglandin E2. 4. It is concluded that activation of TEA-sensitive Ca(2+)-dependent K+ channels does not play a significant role in isoprenaline-induced relaxation. We propose that, in the guinea-pig tracheal muscle, isoprenaline may produce relaxation mainly by inhibiting a receptor-operated pathway for Ca2+ influx across the plasma membrane which is normally activated by prostaglandins.

Focal [Ca2+]i increases detected by aequorin but not by fura-2 in histamine- and caffeine-stimulated swine carotid artery.

1. We hypothesized that the homogeneity of intracellular [Ca2+] ([Ca2+]i) varies and is regulated in arterial smooth muscle. 2. We evaluated this hypothesis by exploiting the different characteristics of several [Ca2+]i indicators: (1) aequorin, which theoretically can measure focal increases in [Ca2+]i, (2) fura-2, which is predominantly a measure of mean cytoplasmic [Ca2+], and (3) myosin light chain phosphorylation and force, which reflect increases in [Ca2+] near the contractile apparatus. 3. From the differences in the observed aequorin and fura-2 signals, we developed an index of the relative degree of [Ca2+]i homogeneity as the ratio of the aequorin signal and fura-2 signal. 4. Stimulation with intermediate concentrations of histamine (1 and 10 microM) or high [K+]o (25 and 40 mM) increased [Ca2+]i and contractile stress. Relative [Ca2+]i homogeneity, estimated from the aequorin/fura-2 ratio, remained similar to levels observed in unstimulated tissues. 5. Higher concentrations of histamine (100 microM) also increased [Ca2+]i and stress, but the aequorin/fura 2 ratio declined , indicating increased [Ca2+]i homogeneity. Similarly, the aequorin/fura-2 ratio decreased when extracellular Ca2+ was removed. 6. Stimulation with histamine in low extracellular [Ca2+] transiently increased [Ca2+]i and the aequorin/fura-2 ratio. Similarly, in tissues treated with low extracellular [Ca2+], restoration of extracellular Ca2+ transiently increased both [Ca2+]i and the aequorin/fura-2 ratio. Although both of these experiments demonstrated a transient decrease in [Ca2+]i homogeneity, only histamine stimulation led to increased myosin light chain phosphorylation and force. These results indicate that the focal increases in [Ca2+]i observed with histamine stimulation and Ca2+ restoration occurred in different cellular regions. 7. Addition of caffeine (20 mM) increased [Ca2+]i and [cAMP], but this was not accompanied by sustained increased myosin light chain phosphorylation or contraction. Phosphorylation of myosin light chain kinase did not appear to underlie the lack of increase in myosin light chain phosphorylation. Rather, caffeine induced a sustained increase in the aequorin/fura-2 ratio, suggesting that caffeine inhibits smooth muscle contraction by localizing increases in [Ca2+]i to a region distant from the contractile apparatus. 8. These data suggest that there can be transient and sustained focal increases in [Ca2+]i. Aequorin detected increased [Ca2+]i in small regions of the cytoplasm during release from and refilling of the intracellular Ca2+ store and with caffeine stimulation. Dual use of aequorin and fura-2 permits determination of relative [Ca2+]i homogeneity in smooth muscle.

Propagation of cytosolic calcium waves into the nuclei of hepatocytes

The temporal and spatial organization of [Ca 2+] changes within the nucleus of Fura-2 loaded hepatocytes maintained in primary culture has been investigated. Vasopressin stimulation induced oscillatory waves of cytosolic free [Ca 2+] increase, which propagated freely through the nuclear region. Based on the amplitude of the Fura-2 signals from this region, the morphology of the hepatocyte nucleus and the rapid penetration of the nucleus by injected Fura-2, it can be concluded that the nuclear Ca 2+ responses reflect changes occurring within the nucleoplasm. Intranuclear Ca 2+ increases occurred as waves that appear to be directed by the Ca 2+ waves passing through the surrounding cytoplasm. The apparent velocity of Ca 2+ waves was higher in the nucleoplasm than in the cytoplasm (19.5 ± 2.9 versus 11.0 ± 1.1 μm/s). The nucleoplasm does not contain vesicular Ca 2+ stores that might be released by Ins(1,4,5)P 3. However, the nuclear envelope functions as a Ca 2+ store that is sensitive to mobilization by Ins(1,4,5)P 3. We conclude that the [Ca 2+] in the nucleoplasm of the hepatocyte is close to equilibrium with the cytosolic [Ca 2+] and that oscillatory waves of cytosolic [Ca 2+] are closely paralleled by similar [Ca 2+] changes in the nucleoplasm. The nuclear envelope is a component of the intracellular Ins(1,4,5)P 3-sensitive Ca 2+ storage pool and may serve as a reservoir for [Ca 2+] elevations within the nucleus.

Calcium influx and release in isolated rat osteoclasts

Intracellular and extracellular sources of cytosolic [Ca2+] elevation in isolated rat osteoclasts were explored by a comparison of fura-2 signals in response to application of the Ca2+ ionophore, ionomycin, in Ca(2+)-containing and in Ca(2+)-free bathing solutions. Cytosolic [Ca2+] transients persisted in osteoclasts bathed in Ca(2+)-free, EGTA-containing solutions. They consisted of a peak cytosolic [Ca2+bd elevation followed by a full decay to baseline and were refractory to manipulations of surface membrane potential through changes in extracellular [K+]. They disappeared upon intracellular Ca2+ store depletion through repeated ionophore applications. They were therefore attributable solely to intracellularly stored Ca2+. In contrast, the fura-2 peaks in osteoclasts exposed to Ca(2+)-containing solutions decayed to sustained levels. Cytosolic [Ca2+] responses then persisted with repeated ionomycin application. These latter phenomena are accordingly attributable to extracellular Ca2+ entry. Finally, restoration of extracellular [Ca2+] to 1.25 mM following the depletion of intracellular Ca2+ stores by treatment with ionomycin elicited a cytosolic [Ca2+] 'overshoot' consistent with capacitative Ca2+ entry via a cytosolic route. These results demonstrate a refillable intracellular source of cytosolic Ca2+ that could function in osteoclastic regulation.

Release of intracellular calcium and modulation of membrane currents by caffeine in bull-frog sympathetic neurones.

1. Calcium release and sequestration were studied in whole-cell voltage-clamped bull-frog sympathetic neurones by image analysis of Fura-2 signals. 2. Application of caffeine (10 mM) to cells voltage clamped at -38 mV caused a rapid increase in intracellular calcium concentration ([Ca2+]i) to a mean value of 352 +/- 33 nM, which activated an outward current. In the continued presence of caffeine the rise in [Ca2+]i slowly declined to a sustained plateau of 196 +/- 20 nM (112 nM above control levels), while the outward current rapidly decayed. Peak calcium release was highest at the edge of the cell. 3. The caffeine-evoked intracellular calcium increase was reduced by two inhibitors of calcium-induced calcium release, ryanodine and procaine. The residual non-suppressible increase in [Ca2+]i may indicate that caffeine can release calcium from two pharmacologically distinct intracellular stores. 4. Inhibition of the caffeine-evoked release of calcium by ryanodine was both concentration and 'use dependent' so that the full inhibitory effect was only observed when caffeine was applied for the second time in the presence of ryanodine. In contrast, the action of procaine did not show any 'use dependence' and unlike ryanodine was fully reversible. 5. The outward current was sensitive to blockers of the large conductance calcium-activated potassium current, Ic. Analysis of variance from this current indicated that it arose at least partly from summation of spontaneous miniature outward currents. 6. The magnitude and duration of calcium release by caffeine was dependent on the resting level of intracellular calcium and the caffeine exposure time. This, together with the pharmacology of the release, suggests that caffeine increases intracellular calcium by sensitizing calcium-induced calcium release. 7. The evoked [Ca2+]i increase was enhanced in amplitude by intracellular application of Ruthenium Red. This effect was mimicked by extracellular application of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) but not by internal application of FCCP or other inhibitors of mitochondrial Ca2+ uptake. This suggests that the evoked increase in [Ca2+]i is predominantly buffered by a Ruthenium Red-sensitive sequestration process which is not mitochondrial.

A model of the single atrial cell: relation between calcium current and calcium release

The hypothesis that calcium release from the sarcoplasmic reticulum in cardiac muscle is induced by rises in free cytosolic calcium (Fabiato 1983, Am. J. Physiol 245) allows the possibility that the release could be at least partly regenerative. There would then be a non-linear relation between calcium current and calcium release. We have investigated this possibility in a single-cell version of the rabbit-atrial model developed by Hilgemann & Noble (1987, Proc. R. Soc. Lond. B 230). The model predicts different voltage ranges of activation for calcium-dependent processes (like the sodium-calcium exchange current, contraction or Fura-2 signals) and the calcium current, in agreement with the experimental results obtained by Earm et al. (1990, Proc. R. Soc. Lond. B 240) on exchange current tails, Cannell et al. (1987, Science, Wash. 238) by using Fura-2 signals, and Fedida et al. (1987, J. Physiol., Lond. 385) and Talo et al. (1988, Biology of isolated adult cardiac myocytes) by using contraction. However, when the Fura-2 concentration is sufficiently high (greater than 200 microM) the activation ranges become very similar as the buffering properties of Fura-2 are sufficient to remove the regenerative effect. It is therefore important to allow for the buffering properties of calcium indicators when investigating the correlation between calcium current and calcium release.

Spontaneous oscillations in cytoplasmic calcium concentration in vascular smooth muscle

Fluorescence measurement of fura-2 and quin2 signals from confluent primary cultures of serum-deprived rat aortic smooth muscle cells have revealed spontaneous oscillations in intracellular calcium concentration ([Ca2+]i). The transients consist of a rapid increase in [Ca2+]i that averages 60 nM and lasts approximately 30 s. They are caused by intracellular calcium release and an influx of extracellular calcium. Exposure of cells to the calcium-channel antagonists verapamil and diltiazem or incubation in nominally calcium-free medium reduced both the duration and amplitude of the transients; in contrast, the calcium-channel agonist (-)BAY K 8644 increased their duration. The transients were abolished by caffeine and 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate, agents that interfere with calcium release from the sarcoplasmic reticulum. These findings demonstrate that the sarcoplasmic reticulum is a primary source for the spontaneous oscillations in cytoplasmic calcium and is closely associated with the influx of extracellular calcium. Although the function of these transients is unclear, they may be involved in the spontaneous contractions observed in some vessels and in the regulation of vascular resistance.

Calcium Currents and Fura-2 Signals in Fluorescence-Activated Cell Sorted Lactotrophs and Somatotrophs of Rat Anterior Pituitary

Optical and electrical recording techniques were applied to single primary pituitary cells to characterize the types of voltage-dependent calcium currents (ICa) and levels of intracellular calcium ([Ca2+]i). GH-containing somatotrophs and PRL-containing lactotrophs were isolated from adult female rats using fluorescence-activated cell-sorting techniques and were maintained in culture for 1-4 days. Whole cell patch-clamp recordings were made to analyze the ICa, and [Ca2+]i was measured with fura-2. Cell type was verified after each recording by indirect immunocytochemistry. GH and PRL cells could be divided into two groups: silent and spontaneously active. Silent cells had stable membrane potentials and stable levels of [Ca2+]i. Spontaneously active cells exhibited spontaneous action potentials and large fluctuations in [Ca2+]i. Two types of ICa were found: a low threshold, transient current which was insensitive to the dihydropyridine -Bay 5417 (the negative isomer of Bay K 8644), and a high threshold, sustained current which was enhanced by -Bay 5417. Both types of ICa were present in PRL and GH cells, but each cell type differed quantitatively in the proportion of each current type. While the GH cells had a more prominent, low threshold, transient ICa, the PRL cells had a more prominent, high threshold, sustained ICa. The enhancement of ICa by -Bay 5417 was greater in the PRL cells, which have a larger dihydropyridine-sensitive ICa. Parallel fura-2 measurements showed an increase in [Ca2+]i in response to 50 mM KCl and -Bay 5417 for both lactotrophs and somatotrophs.

Simultaneous recording of calcium transients in skeletal muscle using high- and low-affinity calcium indicators

To monitor cytosolic [Ca2+] over a wide range of concentrations in functioning skeletal muscle cells, we have used simultaneously the rapid but relatively low affinity calcium indicator antipyrylazo III (AP III) and the slower but higher affinity indicator fura-2 in single frog twitch fibers cut at both ends and voltage clamped with a double vaseline gap system. When both dyes were added to the end pool solution the cytosolic fura-2 concentration reached a steady level equal to the end pool concentration within approximately 2.5 h, a time when the AP III concentration was still increasing. For depolarizing pulses of increasing amplitude, the fura-2 fluorescence signal approached saturation when the simultaneously recorded AP III absorbance change was far from saturation. Comparison of simultaneously recorded fura-2 and AP III signals indicated that the mean values of the on and off rate constants for calcium binding to fura-2 in 18 muscle fibers were 1.49 x 10(8) M-1 s-1 and 11.9 s-1, respectively (mean KD = 89 nM), if all AP III in the fiber is assumed to behave as in calibrating solution and to be in instantaneous equilibrium with [Ca2+]. [Ca2+] transients calculated from the fura-2 signals using these rate constants were consistent with the [Ca2+] transients calculated from the AP III signals. Resting [Ca2+] or small changes in [Ca2+] which could not be reliably monitored with AP III could be monitored with fura-2 with little or no interference from changes in [Mg2+] or from intrinsic signals. The fura-2 signal was also less sensitive to movement artifacts than the AP III signal. After a [Ca2+] transient the fura-2 signal demonstrated a relatively small elevation of [Ca2+] that was maintained for many seconds.